BOC is a Modifier Gene in Holoprosencephaly Mingi Hong1,5, Kshitij Srivastava2,5, Sungjin Kim1, Benjamin L. Allen3, Daniel J. Leahy4, Ping Hu2, Erich Roessler2, Robert S. Krauss1,6,7, and Maximilian Muenke2,6,7 1Department of Cell, Developmental, and Regenerative Biology, Icahn School of Medicine at Mount Sinai, New York, NY, USA 10029. 2Medical Genetics Branch, National Human Genome Research Institute, National Institutes of Health, Bethesda, MD, USA 20892. 3Department of Cell and Developmental Biology, University of Michigan, Ann Arbor, MI, USA, 48109. 4Department of Molecular Biosciences, University of Texas at Austin, Austin, TX, USA, 78712. 5 Contributed equally to this work 6 Contributed equally as senior authors 7Authors for correspondence: Robert S. Krauss (for editorial communication), Department of Cell, Developmental and Regenerative Biology, Icahn School of Medicine at Mount Sinai, New York, NY, 10029, USA. Phone: 212-241-2177; Fax: 212-860-9279 e-mail: [email protected] This is the author manuscript accepted for publication and has undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1002/humu.23286. This article is protected by copyright. All rights reserved. Maximilian Muenke, Medical Genetics Branch, National Human Genome Research Institute, National Institutes of Health, Bethesda, MD, 20892, USA. e-mail: [email protected] Grant sponsors: This work was supported by grants NIH DE024748, GM118751, CA198074; Cancer Prevention and Research Institute of Texas (CPRIT) award RR160023; and by the Division of Intramural Research, NHGRI, NIH. 2 This article is protected by copyright. All rights reserved. Abstract Holoprosencephaly (HPE), a common developmental defect of the forebrain and midface, has a complex etiology. Heterozygous, loss-of-function mutations in the Sonic hedgehog (SHH) pathway are associated with HPE. However, mutation carriers display highly variable clinical presentation, leading to an “autosomal dominant with modifier” model, in which the penetrance and expressivity of a predisposing mutation is graded by genetic or environmental modifiers. Such modifiers have not been identified. Boc encodes a SHH co-receptor and is a silent HPE modifier gene in mice. Here, we report identification of missense BOC variants in HPE patients. Consistent with these alleles functioning as HPE modifiers, individual variant BOC proteins had either loss- or gain-of-function properties in cell-based SHH signaling assays. Therefore, in addition to heterozygous loss-of-function mutations in specific SHH pathway genes and an ill- defined environmental component, our findings identify a third variable in HPE: low frequency modifier genes, BOC being the first identified. KEYWORDS: Holoprosencephaly; Sonic hedgehog; BOC; Modifier gene; Gene variant; Birth defect 3 This article is protected by copyright. All rights reserved. Many structural birth defects are thought to arise from a complex combination of genetic and environmental risk factors (Krauss and Hong, 2016). Holoprosencephaly (HPE; MIM# 236100), a common and often devastating defect in midline patterning of the forebrain and midface, is a prototypical example. HPE encompasses a phenotypic spectrum that ranges from failure to partition the forebrain into hemispheres and cyclopia, to mild midfacial anomalies that occur without forebrain involvement (Geng and Oliver, 2009). Heterozygous, loss-of-function mutations in components of the Sonic hedgehog (SHH; MIM# 600725) signaling pathway are frequently associated with HPE (Roessler and Muenke, 2010). However, highly variable clinical presentation is seen in mutation carriers, even within pedigrees. Furthermore, in many “sporadic” cases, mutations in SHH are inherited from a parent with little or no clinical manifestation (Solomon et al., 2012). Statistical analyses have led to an “autosomal dominant with modifier” model, in which the penetrance and expressivity of a predisposing heterozygous mutation is graded by modifiers (Roessler et al., 2012). Such modifiers may be genetic or environmental in nature. While bona fide pathogenic mutations in SHH and other genes associated with HPE continue to be catalogued, identification of potential HPE modifiers is in its infancy. The binding of SHH to its primary cell surface receptor PTCH1 (MIM# 601309) initiates a signaling cascade to modulate GLI transcription factors, which induce expression of pathway target genes (Lee et al., 2016). Among the direct target genes is GLI1 (MIM# 165220) itself. SHH and PTCH1 both bind to several co-receptors, including CDON (MIM# 608707), BOC (MIM# 608708), and GAS1 (MIM# 139185) (Bae et al., 2011; Izzi et al., 2011; Tenzen et al., 2006). Studies with knockout mice revealed that these three co-receptors have overlapping and partially redundant function in supporting SHH signaling during development; embryos lacking all three have a nearly complete loss of pathway activity (Allen et al., 2011). Heterozygous, loss- of-function mutations in CDON and GAS1 have been identified in HPE patients (Bae et al., 2011; 4 This article is protected by copyright. All rights reserved. Pineda-Alvarez et al., 2012; Ribeiro et al., 2010). Consistent with these observations, mice with targeted mutations in either Cdon or Gas1 display a range of HPE phenotypes (Allen et al., 2007; Hong and Krauss, 2012; Martinelli and Fan, 2007; Seppala et al., 2007; Zhang et al., 2006). In contrast, mice lacking Boc do not have HPE (Zhang et al., 2011). However, genetic removal of Boc from Cdon-/- or Gas1-/- mice exacerbates their HPE phenotype (Seppala et al., 2014; Zhang et al., 2011). Therefore, Boc functions as a silent HPE modifier gene in mice. Here we address the potential role of BOC in HPE pathogenesis in humans. We performed high-throughput screening of BOC (NM_033254.3) for 360 HPE patients using High Resolution Melting, as described (Kauvar et al., 2011). Additionally, 384 unrelated individuals were screened as controls. HPE and control samples with melting profiles that deviated from wild type melting curves were directly sequenced for variant confirmation. A total of eight different amino acid substitution variants in BOC were identified in HPE patients, including one that was found in two unrelated patients and two that were present in a single patient (Table 1). There are over 400 BOC missense variants reported in ExAC. We note that most of the variants we identified are rare (Table 1), including p.Gly556Glu, which has not been previously reported. An exception is p.Pro828Arg, which has a minor allele frequency of 0.0017, more common than the ~1:10,000 birth frequency of HPE (Leoncini et al., 2008), and consistent with possible function as a modifier allele. All individuals were also studied for the four genes most commonly screened in HPE (SHH, ZIC2 (MIM# 603073), SIX3 (MIM# 603714), and TGIF (MIM# 602630)). One patient had a truncating mutation in ZIC2, and another patient had a deletion of TGIF (Table 1). To explore the functional consequences of HPE-associated BOC variants, we developed a cell-based assay for BOC activity. Exogenous expression of BOC had little ability to enhance SHH signaling in wild type mouse embryo fibroblasts (MEFs) or even Cdon-/-;Boc-/- MEFs (not shown). To work in as sensitive a cell system as possible, we used Cdon-/-;Boc-/-;Gas1-/- MEFs 5 This article is protected by copyright. All rights reserved. (TKO cells) (Mathew et al., 2014). When treated with escalating doses of recombinant SHH N- terminus protein (referred to simply as SHH), TKO cells expressed the direct target gene Gli1 very inefficiently, relative to wild type MEFs, as determined by qRT-PCR (Supp. Fig. S1A). To determine whether exogenous expression of CDON or BOC could restore SHH responsiveness to TKO cells, we transfected the cells with expression vectors encoding one or the other of these co-receptors, treated the cultures with SHH, and quantified Gli1 expression. CDON effectively rescued SHH-dependent Gli1 induction, whereas BOC expression had little effect (Supp. Fig. S1B). These results suggest that BOC may require a factor not present in MEFs to act as a productive SHH co-receptor. CDON and GAS1 may play some role in this phenomenon. However, as BOC had little activity in wild type MEFs, and Cdon-/-;Gas1-/- embryos have BOC-dependent SHH activity (Allen et al., 2011; Allen et al., 2007), a need for additional factors for BOC function seems likely. BOC and CDON have Ig repeats followed by fibronectin-type III (Fn3) repeats in their ectodomains (Fig. 1A). The third Fn3 repeat of BOC and CDON bind to SHH, whereas Fn3 repeats 1 and 2 each associate with PTCH1 (Bae et al., 2011; Izzi et al., 2011; Kavran et al., 2010). We constructed expression vectors encoding four of the HPE-associated BOC variants: p.Arg533Leu, p.Gly556Glu, p.Arg697Pro, and p.Pro828Arg. The p.Arg533Leu and p.Gly556Glu substitutions are in Fn3 repeat 1, p.Arg697Pro is in the linker region between Fn3 repeats 1 and 2, and p.Pro828Arg is in the linker region between Fn3 repeat 3 and the transmembrane region (TM). These variants were selected because: 1) they were located in the Fn repeat region of BOC, which is critical for Shh signaling (Song et al., 2015); and 2) they resemble similarly located, HPE-associated, loss-of-function, missense mutations in CDON in that they were highly non- conservative substitutions in evolutionarily conserved residues (Bae et al., 2011). Prioritization of these variants was not based on either retrospective allele frequency or Annovar annotation criteria, which were not available at the time of study design. 6 This article is protected by copyright. All rights reserved. Each variant was expressed at easily detectable levels when transiently transfected in HEK293T cells (Fig. 1B). When expressed in TKO cells subsequently treated with SHH, the p.Arg533Leu, p.Arg697Pro, and p.Pro828Arg variants were similar to wild type BOC in having little ability to promote ligand-dependent Gli1 expression (Fig.